Protein Kinase Inhibitor H7 Research Articles

Protein Kinase Inhibitor H7 Blocks the Induction of Immediate-Early Genes zif268 and c-fosby a Mechanism Unrelated to Inhibition of Protein Kinase C but Possibly Related to Inhibition of Phosphorylation of RNA Polymerase II

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes, zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922-23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

Use-Dependent Decline of Paired-Pulse Facilitation atAplysiaSensory Neuron Synapses Suggests a Distinct Vesicle Pool or Release Mechanism

We have characterized paired-pulse facilitation at Aplysia sensory neuron-to-motoneuron synapses. This simple form of very short-term synaptic plasticity displayed an unusual feature: it decreased dramatically with repeated testing. Synaptic depression at these synapses and this use-dependent decrease in paired-pulse facilitation occurred independently of each other. Paired-pulse facilitation was inversely correlated with the size of the initial synaptic connection and was absent at stronger synapses. The use-dependent decrease in paired-pulse facilitation occurred at the same rate at large synapses as at small synapses, although the initial paired-pulse facilitation at large synapses was substantially smaller. Rates of synaptic depression were also independent of initial synaptic strength. Paired-pulse facilitation was blocked by presynaptic EGTA injection, but not by postsynaptic EGTA or BAPTA injection. These results indicate that presynaptic Ca2+ influx plays a critical role in paired-pulse facilitation. However, the persistence of the decrease in paired-pulse facilitation for longer than 15 min suggests that Ca2+ from the first paired action potential produces facilitation via a modulatory mechanism rather than by summating with Ca2+ influx during the second paired action potential in activating the Ca2+ binding sites that initiate exocytosis. This modulatory mechanism may not involve protein phosphorylation because paired-pulse facilitation was unaffected by the protein kinase inhibitors H7 and KN-62. These findings further suggest that release by the second paired action potential occurs at sites distinct from those that mediate release by the first action potential.

The inhibitory effect of beta-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway.

The beta-agonist isoproterenol (ISO) reduces the Na/K pump current (Ip) via beta-adrenergic receptors when the intracellular calcium concentration ([Ca2+]i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 microM), or the adenylyl cyclase activator forskolin (50 microM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 microM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on Ip is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H7 (100 microM) nor the protein phosphatase inhibitor calyculin A (0.5 microM) had any effect on Ip in the absence of ISO. However, H7 could increase Ip and calyculin A could reduce it in the presence of ISO (1 microM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.

Substance P modulates sensory action potentials in the lamprey via a protein kinase C-mediated reduction of a 4-aminopyridine-sensitive potassium conductance.

We have examined the effects of the tachykinin substance P on the action potential of lamprey mechanosensory dorsal cells. Substance P increased the spike duration and reduced the afterhyperpolarization. These effects were mimicked by stimulation of the dorsal root, which contains tachykinin-like immunoreactive fibres. The tachykinin antagonist spantide II blocked the effects of both substance P and dorsal root stimulation. The spike broadening was voltage-dependent, and was due to the reduction of a 4-aminopyridine-sensitive potassium conductance. The spike broadening was mimicked by G-protein activators and blocked by the G-protein inhibitor GDPbetaS. Pertussis toxin did not block the effects of substance P. The spike broadening was blocked by the protein kinase C and cAMP-dependent protein kinase inhibitor H7, and by the specific protein kinase C antagonist chelerythrine, but not by the cAMP and cGMP-dependent protein kinase inhibitor H8. The phorbol ester phorbol 12,13-dibutyrate mimicked and blocked the effects of substance P, supporting the role of protein kinase C in the spike modulation. The adenylate cyclase activator forskolin and the cAMP agonist SpcAMPs mimicked but did not block the effects of substance P on the spike duration, suggesting that protein kinase A also modulates the dorsal cell action potential, but that substance P acts independently of this pathway. Substance P also increased the excitability of the dorsal cells. This effect was blocked by 4-AP, PDBu and chelerythrine, but not by H8, suggesting that the increase in excitability shares the same intracellular and effector pathways as the spike broadening.

A Postsynaptic Interaction between Dopamine D1and NMDA Receptors Promotes Presynaptic Inhibition in the Rat Nucleus Accumbens via Adenosine Release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.

Tolerance to mu-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5'-O-(3-[35S]-thio)triphosphate binding.

1. The agonist action of morphine on membranes prepared from human neuroblastoma SH-SY5Y cells was measured by an increase in the binding of the GTP analogue [35S]-GTPgammaS. Morphine increased the binding of [35S]-GTPgammaS to SH-SY5Y cell membranes by 30 fmol mg(-1) protein with an EC50 value of 76 +/- 10 nM. 2. Incubation of SH-SY5Y cells with 10 microM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 value with a 31 +/- 6% decrease in the maximum stimulation of [35S]-GTPgammaS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. 3. Chronic treatment of the cells with the more efficacious mu-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO, 10 microM) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9 +/- 6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8 +/- 4%. There was a complete loss of agonist activity for pentazocine. 4. The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative beta-adrenoceptor kinase (beta-ARK) inhibitor suramin. 5. The observed tolerance effect was accompanied by a down-regulation of mu-opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [3H]-diprenorphine of 66 +/- 4%, but with no change in binding affinity. Binding of the agonist [3H]-DAMGO was similarly reduced. 6. The modulation of [35S]-GTPgammaS binding in SH-SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.

Modulation of ligand-gated dopamine channels in Helix neurones.

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O-permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of 32P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) 32P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12-O-tetradecanoylphorbol 13-acetate and 1,2-dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.

Intra-Ventral Tegmental Area Administration of H7 Delays, But Does Not Prevent the Development of Cocaine-Induced Sensitization

Previous studies have suggested that increased protein kinase C activity in the ventral tegmental area (VTA) may play a role in the acute and development of the sensitized behavioral responses to cocaine. The present study was conducted to further characterize the role of protein kinases in the development of sensitization. Animals received injections of saline or the nonspecific protein kinase inhibitor H7 into the VTA before each of their four daily systemic injections of saline or cocaine. Animals were tested for sensitization with a challenge injection of systemic cocaine after a withdrawal period of 24 h or 1 week. Tests for sensitization included monitoring cocaine-induced motor activity and/or dopamine concentrations in the nucleus accumbens, as measured by in vivo microdialysis. Pretreatment with H7 in the VTA attenuated the acute motor stimulant response to cocaine as well as the cocaine-induced increase in extracellular dopamine in the nucleus accumbens. In addition, the augmented increase in dopamine in the nucleus accumbens of cocaine-sensitized animals was prevented in animals pretreated with H7 before each of their daily cocaine injections, when tested after a 24 h withdrawal. However, when tested after a 1 week withdrawal, animals demonstrated sensitization to both the cocaine-induced increase in motor activity and the cocaine-induced increase in dopamine in the nucleus accumbens regardless of whether they received intra-VTA saline or H7 before each of their daily cocaine injections. These data suggest that injection of a protein kinase inhibitor into the VTA delays, but does not prevent the development of cocaine-induced behavioral sensitization.

Crystal Structures of Catalytic Subunit of cAMP-dependent Protein Kinase in Complex with Isoquinolinesulfonyl Protein Kinase Inhibitors H7, H8, and H89: STRUCTURAL IMPLICATIONS FOR SELECTIVITY

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.

2,3-Butanedione monoxime modifies the glycine-gated chloride current of acutely isolated murine hypothalamic neurons

In this study, we explored the effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on glycine current ( I Gly of murine ventromedial hypothalamic neurons. Co-application of 0.01 to 67 mM BDM increased I Gly decay rate with little change of the peak amplitude. This effect was both rapid in onset and offset and required the presence of the agonist. Pretreatment with BDM alone did not alter I Gly decay. In addition, dialysis of neurons with 500 μM ATP-γ-S did not alter the acute effect of BDM. Thus, this effect may result from open channel block rather than BDM-induced dephosphorylation of the receptor/channel protein. In contrast to the acute effect described above, relatively prolonged (i.e., greater than 80 s) pretreatment with BDM reduced peak I Gly The phorbol ester (PDBu), a protein kinase C (PKC) activator, mimicked this effect of BDM. Furthermore, chelerythrine, a specific PKC inhibitor, prevented this effect of BDM on peak I Gly Thus, activation of PKC may mediate this attenuating effect of BDM on I Gly For a sub-population of these pretreated neurons, there was a subsequent potentiation of I Gly which followed the initial suppressant effect. This potentiation may be due to a phosphatase effect of BDM, since it was observed more frequently when neurons were also pretreated with the protein kinase inhibitors H7 or chelerythrine. These findings suggest that BDM alters protein kinase activity and acts as a phosphatase to regulate the activity of the glycine receptor/channel complex.

Interactions between cAMP- and cGMP-dependent protein kinase inhibitors and phosphodiesterase IV inhibitors on arachidonate release from human monocytes

The effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type IV inhibitors and of the cell permeable analogue of cAMP, db-cAMP, were investigated on fMLP-induced arachidonate release from human monocytes. When monocytes were preincubated with the combined PKA/PKG inhibitor H8 (10(-6) to 10(-4) M) or the selective PKG inhibitor Rp-8-cpt-cGMPs (10(-6) to 10(-4) M) a concentration-dependent reduction of the inhibitory effect of db-cAMP (10(-3) M), rolipram (10(-5) M) and Ro 20-1724 (10(-5) M) was noted. When monocytes were preincubated with the selective PKA inhibitor H89 (10(-6) to 10(-4) M), only a small inhibition of the effect of db-cAMP and no inhibition of the effects of rolipram and Ro 20-1724 were observed. The present data indicate that db-cAMP and PDE IV inhibitors elicit an in vitro anti-inflammatory activity by a PKA-independent mechanism, which do not appear to be mainly mediated via the PKG activation.

Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes.

The ability of cAMP to modulate the actions of Ca(2+)-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca(2+)-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca(2+)-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.

A cardiotonic steroid bufalin-induced differentiation of THP-1 cells: Involvement of NA +, K +-ATPase inhibition in the early changes in proto-oncogene expression

Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na +, K +-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1β (IL-1β). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto-oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to inhibit bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na + was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na + medium. These findings indicate that an increased intracellular Na + concentration resulting from the Na +, K +-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.

Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurones from adult rats.

Capsaicin activates a non-specific cation conductance in mammalian sensory neurones. If capsaicin is applied continuously or repeatedly then there is a progressive decline in responsiveness. We have studied the mechanism of this desensitization using electrophysiological methods in cultured dorsal root ganglion neurones from adult rats. The rate of desensitization of capsaicin-induced responses is partly dependent on the extracellular calcium concentration and is slower when extracellular calcium is reduced. Desensitization is strongly inhibited by intracellular application of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid (BAPTA). These data suggest that desensitization is due to a rapid rise in intracellular calcium levels which occurs when capsaicin-sensitive ion channels are activated. Desensitization is not reduced by the non-specific protein kinase inhibitors H7 or staurosporine or by okadaic acid, a selective inhibitor of protein phosphatases 1 and 2A. Desensitization is greatly reduced by cyclosporin A complexed to cyclophilin, which is a specific inhibitor of protein phoshatase 2B (calcineurin). A mechanism for desensitization of capsaicin responsiveness is proposed whereby the evoked rise in calcium activates calcineurin leading to dephosphorylation and desensitization of the capsaicin-sensitive ion channels.

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source.

Phosphoenolpyruvate (PEP) carboxylation was measured as dark 14CO2 fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO2 fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO2-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO2 fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4-5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.

Neurotensin induces calcium oscillations in cultured amacrine cells.

The peptide, neurotensin, is found in a class of amacrine cells synapsing chiefly with other amacrine cells in the chicken retina (Li & Lam, 1990; Watt et al., 1991). To investigate the possible effects of neurotensin, we have used Ca2+ imaging to measure cytosolic Ca2+ concentrations in cultured chick amacrine cells. Following a delay of about 2 min, neurotensin (300 nM) induced oscillations in Ca2+ concentration that typically had a period of 2 min and peak values of about 300 nM when averaged over the cell body. The phospholipase C inhibitors U-73, 112 and 4'-bromophenacyl bromide terminated oscillations induced by neurotensin but the protein kinase inhibitors H7 and staurosporine did not inhibit oscillations, increasing their frequency instead. In the absence of external Ca2+, neurotensin induced only a single Ca2+ transient, much briefer than when external Ca2+ was present. Together these results suggest that neurotensin activates phospholipase C, thereby producing IP3 that triggers Ca2+ release from an internal store. Although this released Ca2+ contributes to periodic Ca2+ peaks, the majority of cytosolic Ca2+, even in the first peak, comes from Ca2+ influx across the plasmalemma.

Forskolin Stimulates Aggrecan Gene Expression in Cultured Bovine Chondrocytes.

Prostaglandins are autacoids that elevate intracellular 3prime prime or minute:5prime prime or minute-cyclic adenosine monophosphate (cAMP) levels in chondrocytes and other cells in culture. To facilitate intracellular cAMP accumulation, bovine chondrocytes were incubated with forskolin alone or forskolin and isobutylmethylxanthine. Both significantly increased proteoglycan synthesis, which was inhibited by the cAMP-dependent protein kinase inhibitor H89. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on 3--16% gels revealed the presence of two large proteoglycan core proteins which migrated more slowly than the 200-kDa marker protein and two small proteoglycan core proteins which migrated slightly slower than the 46-kDa marker. Northern blot hybridization, employing (32)P-labeled cDNA probes, showed that aggrecan steady-state mRNA levels were increased by forskolin and isobutylmethylxanthine after 1 h and 5 h incubation. Decorin and type II collagen mRNA levels were not altered under these conditions. Link protein mRNA levels were slightly elevated, but only at the 5-h time point. These results indicated that stimulation of intracellular cAMP accumulation by forskolin or forskolin and isobutylmethylxanthine resulted in augmented proteoglycan synthesis via increased steady-state aggrecan mRNA levels. Suppression of proteoglycan synthesis by the cAMP-dependent protein kinase inhibitor H89 suggested that cAMP-dependent protein kinase may also play a role in regulating the synthesis and completion of newly synthesized proteoglycans.

Thyroid Hormones Stabilize Acetylcholinesterase mRNA in Neuro-2A Cells That Overexpress the β1 Thyroid Receptor

We investigated the intracellular events involved in the 3,3',5-triiodo-L-thyronine (T3)-induced accumulation in acetylcholinesterase (AChE) activity in neuroblastoma cells (neuro-2a) that overexpress the human thyroid receptor beta 1 (hTR beta 1). Treatment of these cells with T3 increased AChE activity and its mRNAs after a lag period of 24-48 h, and these levels increased through stabilization of the transcripts by T3. T3 had no effect on the transcriptional rate or processing of AChE transcripts. The protein kinase inhibitor H7 inhibited T3-induced accumulation in AChE activity and its mRNAs, whereas okadaic acid (a potent inhibitor of phosphatases 1 and 2A) potentiated the effect of T3. Okadaic acid and H7 have no effect on the binding of hTR beta 1 to T3 or the transcriptional rate of the AChE gene. Finally, treatment of cells with T3 stimulated cytosolic serine/threonine, but not tyrosine kinase, activities. The time course analysis reveals that the increase in serine/threonine activity precedes the effect of T3 on AChE mRNAs. These results suggest that activation of a serine/threonine protein kinase pathway might be a link between nuclear thyroid hormone receptor activation and stabilization of AChE mRNA.